J. Conde

Oxidative Stress Triggers STAT3 Tyrosine Phosphorylation and Nuclear Translocation in Human Lymphocytes

Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H2O2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with H2O2. Moreover, anti-STAT3 antibodies specifically precipitated a protein of 92 kDa that becomes phosphorylated on tyrosine upon lymphocyte treatment with H2O2. Phenylarsine oxide, a tyrosine phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor, cooperated and cancelled, respectively, the H2O2-promoted STAT3 nuclear translocation. Evidence is also presented, using Fe2+/Cu2+ions, that ⋅OH generated from H2O2through Fenton reactions could be a candidate oxygen reactive species to directly activate STAT3. Present data suggest that H2O2 and vanadate are likely to inhibit the activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the nucleus. These results demonstrate that the DNA binding activity of STAT3 can be modulated by oxidizing agents and provide a framework to understand the effects of oxidative stress on the JAK-STAT signaling pathway.

Characterization of Calcineurin in Human Neutrophils INHIBITORY EFFECT OF HYDROGEN PEROXIDE ON ITS ENZYME ACTIVITY AND ON NF-κB DNA BINDING

We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-κB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-κB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-κB inactivation in neutrophils occurs in concert with inhibition of calcineurin.

The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen

Many works have dealt with the study of the allergenic relevance of profilin from allergenic extracts, mainly derived from pollens and vegetable foods. Olive pollen extracts also contain a profilin allergen (Ole e 2). This protein has been characterized in detail, so the aminoacid sequence of three isoforms and the structural model of one of them are already known. The prevalence of Ole e 2 for olive allergenic patients has been evaluated by different in vivo and in vitro methods, and the results compared with those obtained for another pollen profilins.

Prevalence of sensitization to Tetranychus urticae in greenhouse workers.

Tetranychus urticae (TU) is a macroscopic mite which infests a large number of plants of economic interest worldwide. It has recently been described as a cause of occupational allergic disease in greenhouse workers. However, there are no epidemiological data concerning the prevalence of TU allergy in an unselected exposed population.

A sensitive monoclonal antibody‐based enzyme‐linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2

Background Parietaria pollen is one of the most important causes of pollinosis in Mediterranean countries. Parietaria judaica pollen extract presents two major allergens, Par j 1 and Par j 2, that belong to the lipid transfer protein family.

Seizures induced by NSAID.

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l‐Selectin expression on neutrophils from allergic patients

Background L‐selectin (CD62L) is an adhesion molecule involved in leucocyte attachment to endothelium at sites of inflammation, and it has been demonstrated that L‐selectin is rapidly shed after neutrophil activation. Recently, it has been reported that there is increasing evidence of neutrophil participation in asthma and the allergic process.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1.8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Development of a sandwich-type ELISA for measuring Pla a 1, the major allergen of Platanus acerifolia pollen.

Background:Platanus acerifolia is an important cause of pollinosis in Western European cities. Pla a 1, a nonglycosylated 18-kDa protein with a prevalence of 80%, is a major allergen in P. acerifolia pollen extracts. Our aim was to develop a Pla a 1-specific ELISA to quantify this protein in allergenic extracts and preparations for clinical use. Methods: Pla a 1 was purified by cation exchange at pH 7.0, gel filtration, and anion exchange chromatography at pH 10.0. Monoclonal (mAb) and polyclonal antibodies were obtained by immunizing mice and rabbits with nPla a 1. One (5C1) of the 13 mAb obtained was used as capture antibody at 5 µg/ml and biotin-labeled specific polyclonal antiserum at 0.63 µg/ml served for detection. Results: The prevalence of Pla a 1-specific IgE to purified Pla a 1 among 47 P. acerifolia-allergic patients was 79%. The Pla a 1-ELISA developed has a linear range of 3–25 ng/ml, high sensitivity with a detection limit of 0.5 ng/ml and is highly specific as none of the 24 pollen, mite, mold, and plant food extracts tested gave positive results. The assay could quantify Pla a 1-like proteins in other planetree pollen extracts. A good correlation was obtained between Pla a 1 content of 11 P. acerifolia pollen extracts (average content 0.69% of the total protein) and their IgE-binding activity. Conclusions: The described two-site sandwich ELISA to measure Pla a 1 is useful for standardization of planetree pollen extracts intended for clinical use.

Peripheral blood T lymphocytes in seasonal bronchial asthma.

Variations in T lymphocytes in asthmatic patients are related to disease severity. However, the effects of natural exposure to pollens on peripheral blood T lymphocytes have not been clarified. In this paper, the effects on peripheral blood CD4 and CD8 lymphocytes from pollen‐sensitive subjects and from nonatopic donors were studied during and outside the pollen season. In patients who suffer from seasonal asthma, we found an increase in the CD4/CD8 bright ratio and a decrease in the mean number of CD4 receptors per cell during the pollen season. No variation was observed in healthy subjects. These results suggest that CD4 lymphocytes may be causally linked to the pathogenesis of seasonal bronchial asthma.